Tumor cell metastasis is the leading cause of mortality in breast cancer patients. Cancer cells invade surrounding tissue and blood vessels by forming filamentous- (F-) actin-rich protrusions called invadopodia that degrade the extracellular matrix (ECM). A major challenge in breast cancer research is the elucidation of the mechanisms that underlie invadopodia formation. Cortactin, an actin-binding protein, nucleates invadopodia formation and promotes breast cancer cell metastasis. These properties of cortactin are potentiated by its tyrosine phosphorylation. We have shown that adhesion to ECM activates integrin adhesion receptors to stimulate the Arg tyrosine kinase. In turn, Arg phosphorylates cortactin to promote dynamic invadopodia-like cell edge protrusions. Integrins [unreadable]1 and [unreadable]3, and Arg are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function in these cells compromises their invasiveness. Our proposal will elucidate the mechanisms by which the integrin-Arg-cortactin axis controls invadopodia formation and function. Our first aim is to test whether integrins [unreadable]1 and [unreadable]3 signal through Arg and cortactin to regulate invadopodia formation, ECM invasiveness, and tumor metastasis. We will determine whether ECM adhesion stimulates cortactin phosphorylation in breast cancer cells with varying degrees of invasiveness and test if knockdown of integrins [unreadable]1 or [unreadable]3, or Arg compromise this process. We will use tissue culture assays to determine whether knockdown/knockout of integrins [unreadable]1 or [unreadable]3, Arg, or cortactin disrusions. Integrins [unreadable]1 and [unreadable]3, and Arg are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function in these cells compromises their invasiveness. Our proposal will elucidate the mechanisms by which the integrin-Arg-cortactin axis controls invadopodia formation and function. Our first aim is to test whether integrins [unreadable]1 and [unreadable]3 signal through Arg and cortactin to regulate invadopodia formation, ECM invasiveness, and tumor metastasis. We will determine whether ECM adhesion stimulates cortactrich protrusions called invadopodia that degrade the extracellular matrix (ECM). A major challenge in breast cancer research has been to elucidate the mechanisms that underlie invadopodium formation. Cortactin, an actin-binding protein, nucleates invadopodia formation and promotes breast cancer cell metastasis. These properties of cortactin are potentiated by its tyrosine phosphorylation. Adhesion of fibroblasts to ECM activates integrin adhesion receptors to stimulate the Arg tyrosine kinase. In turn, Arg phosphorylates cortactin to promote dynamic invadopodia-like cell edge protrusions. Interestingly, integrins [unreadable]1 and [unreadable]3, Arg, and cortactin are all upregulated in highly invasive breast cancer cells, and inhibiting Arg or cortactin function compromises their invasiveness. Utilizing our expertise and that of our collaborators, we are in a unique position to delineate the relevance of the integrin-Arg-cortactin signaling axis in determining breast cancer invasiveness and metastasis via invadopodia formation. We have three specific aims: Aim 2. To understand how Arg interacts with cortactin to promote actin polymerization in invadopodia. Cortactin stimulates the ability of the Actin-related protein 2/3 (Arp2/3) complex to promote actin filament nucleation and also stabilizes the resulting branched F-actin networks. However, it remains unclear how these activities of cortactin are regulated to promote invadopodium formation. Our preliminary studies provide evidence that Arg binding to and phosphorylation of cortactin regulates cortactin's ability to promote F-actin protrusions. We will determine the biochemical mechanisms by which Arg regulates cortactin by: A. Mapping the interaction domains in Arg and cortactin. Arg binds to cortactin via at least two distinct sets of protein:protein interactions. We will map which surfaces in Arg and cortactin mediate these interactions and test how disruption of these surfaces affects Arg:cortactin interactions. B. Determining the significance of Arg-cortactin interactions in F-actin nucleation. We will test whether Arg binding to and phosphorylation of cortactin affect its ability to stimulate actin filament nucleation by the Arp2/3 complex and promote actin branch stability. Aim 3. To understand how cortactin phosphorylation regulates invadopodium formation/function. In a large-scale screen of nearly all (100 out of 115) Src homology 2 (SH2) domains in the human proteome, we have identified a small subset that bind selectively phospho-cortactin (cortactin-P). We hypothesize that these proteins synergize with cortactin-P to promote assembly and stability of the invadopodium F-actin core. We will determine how these proteins regulate cortactin's ability to promote invadopodium formation/function by: A. Testing requirement of phosphorylation sites in invadopodium formation. Arg phosphorylates cortactin on three tyrosine residues (Y421, Y466, Y482). We will use Y421, Y466, and Y482 cortactin point mutants to identify essential phosphorylation sites required for invadopodium formation. B. Measuring affinity and specificity of cortactin-P interacting proteins. We will determine the specificity of individual SH2 domains for the phosphotyrosine residues in cortactin using the same set of mutants. C. Characterizing roles for cortactin-P-binding proteins in invadopodium formation. We will use live cell imaging and RNAi-mediated protein knockdown to examine whether the cortactin-P-binding proteins localize to invadopodia and regulate their formation.